The mouse mammary tumor virus (MMTV) long terminal repeat (LTR) contains the target site for steroid hormone action associated with this virus. In order to directly address the molecular mechanisms by which hormone-receptor complex, RNA polymerase II and other nuclear protein components interact with the MMTV LTR to produce the observed biological phenomenon of elevated rates of transcription, we think that reconstitution of hormone-regulated transcription in vitro will be required. We have previously introduced MMTV LTR into eukaryotic cells as an episome, by using the bovine papillomavirus (BPV) as a vector. The MMTV LTR, present at 200 copies per cell, is still responsive to steroid-specific induction of transcription in this episomal form. The episomal elements can be extensively purified from cellular chromatin by selective extraction from the nuclear fraction, sedimentation through sucrose gradients, and concentration. The purified minichromosomes retain their association with 2 to 6 moles of glucocorticoid receptor subunit; this association is specific in that no bound receptor is found with control episomes not containing the MMTV LTR sequences. The fraction is highly enriched, with greater than 90% of the DNA in the fraction of episomal origin. These minichromosomes should serve as useful templates for in vitro transcription studies.